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  Glial and vascular contributions to neurodegenerative diseases
Blood-brain barrier leakage in cerebral amyloid angiopathy
Sabine Voigt1, K Kaushik1, M R Schipper1, R G J van der Zwet1, R van Dort1, J J de Jong2, W H Backes2, S J van Veluw1,3,4, E I Hoff5, J Staals2, M J P van Osch1, M A A van Walderveen1, M J H Wermer1, W M Freeze1
1Leiden University Medical Center, Leiden, The Netherlands, 2Maastricht University Medical Center, Maastricht, The Netherlands, 3J. Philip Kistler Stroke Research Center, Boston, USA, 4MassGeneral Institute for Neurodegenerative Disease, Charlestown, USA, 5Zuyderland Medical Center, Heerlen, The Netherlands
 Background: Mechanisms underlying hemorrhagic lesion formation in cerebral amyloid angiopathy (CAA) remain incompletely understood. Pre-clinical studies suggest that vessel wall breakdown may be part of a vicious cycle, including amyloid-beta accumulation, vascular dysfunction, impaired perivascular clearance, and vascular remodeling, eventually resulting in vessel rupture. Our aim is to establish whether BBB leakage, as a marker of vessel wall breakdown in CAA, is associated with hemorrhagic brain injury in CAA.
Materials and methods: In this prospective cross-sectional study, we aim to include 25 participants with probable CAA without prior intracerebral hemorrhage from the ongoing FOCAS natural history study, and 20 age- and sex matched non-neurological control cases. We are performing a 3 Tesla MRI scan, neuropsychological assessment, blood withdrawal and blood pressure measurement. Parenchymal BBB leakage severity and location are determined on gadolinium-enhanced T1-weighted DCE-MRI[1]. Leptomeningeal BBB leakage is assessed on heavily T2-weighted post-contrast FLAIR images[2]. Associations between BBB leakage and cerebral microbleed burden and cortical superficial siderosis will be investigated.
Results: In the first two participants with probable CAA (man of 63 years and woman of 78 years) we found BBB leakage rates (Ki) of 6.7 and 9.4*10-6 min-1 in the cortex, and BBB leakage rates of 3.3 and 8.2*10-6 min-1 in the white matter. In one of these patients we found a focus of leptomeningeal BBB leakage in the occipital lobe. No control participants have been scanned yet.
Discussion: Based on the current sample size of n=2 it is not possible to determine whether BBB leakage is associated with CAA and hemorrhagic brain pathology. We expect that by the end of summer 2022 we will have scanned n=10 probable CAA patients and n=10 control participants.
Conclusion: Our preliminary results suggest that it is possible to evaluate BBB leakage in CAA patients with DCE-MRI and post-contrast FLAIR MRI.
Background: Cerebral small vessel disease (SVD) is the major vascular contributor to cognitive decline leading to vascular dementia. CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), is the leading heritable SVD caused by mutations in the NOTCH3 gene that is predominantly expressed in vascular smooth muscle cells (VSMCs) and pericytes. Aberrant NOTCH3 extracellular domain (NOTCH3 ECD) deposit around VSMC is the diagnostic hallmark of CADASIL. Autophagy, a key cellular self- degradative process important for maintaining proteostasis, is impaired in most neurodegenerative diseases. Our aim is to elucidate how autophagy impacts the CADASIL pathology and progression caused
by NOTCH3 mutation by using CADASIL mouse models with impaired autophagy.
Methods: CADASIL mice are crossed with autophagy-deficient mice of various promoters using Cre-loxP system to investigate the role of autophagy in the onset and progression of CADASIL. The workflow includes immunohistochemistry analysis of mouse brain slices and retina wholemounts, measurement of circulating NOTCH3 ECD in serum using ELISA, as well as cerebral vessel isolation and purification for subsequent studies.
Results: The presence of purified cerebral small vessels was confirmed using immunofluorescence (IF) and Western blot staining with various vasculature and neuronal markers. In the Atg7f/f;CaMK II-Cre mouse model, p62 aggregation was observed by IF staining and Western blot. The circulating concentration of NOTCH3 ECD in the serum of CADASIL mouse model is higher than in the control mouse. We discovered that NOTCH3 ECD aggregates around cerebral small vessels in the CADASIL mouse model using mouse brain sections and purified microvessels. Conclusions: The protocol we used to isolate cerebral small vessels allows us to perform proteomic and transcriptomic analysis using various CADASIL mouse models with impaired autophagy. Further studies will shed light on how the autophagic system functions in CADASIL disease.
Insights into the mechanisms and factors initiating pathologic consequences in microvessels after neural injury
Tian Zhou1, Yiming Zheng1, Grace Hammel1, Li Sun1, Smaranda Ruxandra Badea2, Haitao Sun2, Choogon Li1, Timothy Megraw1, Wutian Wu2, Yi Ren1
1Florida State University College of Medicine, TALLAHASSEE, USA, 2School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Background: Dysfunction of the endothelium lining of blood vessels is a hallmark characteristic of various types of neural injury and neuroinflammatory disorders and is typically characterized by endothelial cell (EC) activation, loss of endothelium integrity of the blood-spinal cord-barrier (BSCB), and enhanced inflammation. However, the entire spectrum of pathologic consequences that arise from EC dysfunction, as well as the mediators of these consequences, lack a complete understanding.
Materials and Methods: The mouse models of spinal cord injury (SCI) and multiple sclerosis (MS) were used in the study. The transendothelial FITC-dextran permeability analysis and transendothelial electrical resistance measurements (TEER) were used to quantify endothelial barrier integrity.
Results: We demonstrated that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further showed that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating neuroinflammation, pathologic angiogenesis and fibrosis in both SCI and MS. Furthermore, we found the downregulation of gene expression associated with endothelial BSCB cell-to-cell junctions. We further explored the role of myelin debris as a crucial neural lesion related factor that directly participates in induction of BSCB disruption in the acute injured spinal cord. We showed that myelin debris engulfment not only significantly decreases VE-cadherin protein levels in ECs but also disrupts another key junctional protein (ZO-2) in ECs. We also assessed the permeability of myelin debris-laden ECs (Mye-ECs) and found that Mye-ECs have a significantly decreased barrier integrity, and significantly lower TEER readings.
Conclusions: These results suggest that myelin debris can act as a pathological mediator in ECs after neural injury. Therefore, targeting myelin debris engulfment by ECs may represent a novel therapeutic approach for neural injury.
Involvement of autophagy in small vessel disease using CADASIL Notch3 disease animal models
Wenchao Shao1
1Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Solna, Sweden

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